CN115010720B - Chinese mugwort sesquiterpene dimer and pharmaceutical composition thereof, and preparation method and application thereof - Google Patents
Chinese mugwort sesquiterpene dimer and pharmaceutical composition thereof, and preparation method and application thereof Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/10—Spiro-condensed systems
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Abstract
The invention provides a sesquiterpene dimer in Chinese mugwort, a pharmaceutical composition thereof, a preparation method and application thereof, and belongs to the technical field of medicines. The 21 novel sesquiterpene dimers shown in the structural formula (I) have obvious cytotoxic activity on human hepatic stellate cells (HSC-LX 2) by artemzhongdianolides A-A21 (1-21), can form a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient, and can be used for preparing anti-hepatic fibrosis drugs.
Description
Technical field:
the invention belongs to the technical field of medicines. In particular to a sesquiterpene dimer compound artemzhongdianolides A-A21 (1-21), a preparation method, a pharmaceutical composition and application thereof.
The background technology is as follows:
liver fibrosis is a common pathological process of most liver diseases, namely excessive deposition of extracellular matrix (extracellular matrix, ECM) in the liver under the action of various pathogenic factors, thereby affecting the physiological structure and function of the liver, and if the liver is not inhibited or reversed in time, the occurrence of liver cirrhosis or liver cancer can be accelerated, and the life quality of a human body is seriously affected. At present, single control of etiology or liver protection by traditional Chinese medicines is a main mode for treating liver fibrosis, and no chemical synthetic medicine aiming at the treatment of liver fibrosis is clinically available. Although development of anti-fibrosis drugs has progressed to some extent in recent years, some candidate drugs have difficulty in achieving the intended therapeutic effect or occurrence of side effects in clinical trials. Hepatic stellate cells (hepatic stellate cells, HSCs) are the primary source of myofibroblasts. In normal liver, HSCs proliferate very slowly, which maintains their homeostasis by regulating extracellular matrix synthesis and degradation. When the liver is continuously stimulated by external pathogenic factors, a series of complex mechanisms are started, and the end result is that HSC is converted from resting type to activated type, various fibroblast growth factors such as TGF-beta, PDGF and the like are secreted, a large amount of unordered extracellular matrix (extracellular matrix, ECM) is generated, and the occurrence and development of fibrosis are promoted. Therefore, HSC is a potential target for anti-hepatic fibrosis treatment, and many anti-hepatic fibrosis drugs are currently being studied to achieve therapeutic purposes by inhibiting HSC proliferation and fibrogenesis, promoting HSC apoptosis, and the like. The present invention aims to find new anti-hepatic fibrosis active ingredients from natural products based on a human hepatic stellate cell (hepatic stellate cells line LX, HSC-LX 2) screening model. The Artemisia princeps (Artemisia zhongdianensis Y.R.Ling) belongs to Artemisia of Compositae, is perennial herb, and is distributed in the northwest part of Yunnan (Zhongdian).
To date, the prior art has no report of sesquiterpene dimer artemzhongdianolides A-A21 (1-21), nor of compound 1-21 and its pharmaceutical composition as anti-liver fibrosis drug.
The invention comprises the following steps:
the invention aims to provide a novel sesquiterpene dimer with medicinal value, artemzhongdianolides A-A21 (1-21), a preparation method and application thereof, a pharmaceutical composition containing artemzhongdianolides A-A21 and application thereof, and in the early work of the invention, the middling Ai Yichun extract has remarkable HSC-LX2 cytotoxicity (the inhibition rate reaches 85.7% at the concentration of 400 mug/mL), 21 novel sesquiterpene dimers are obtained through activity-oriented separation, artemzhongdianolides A1-A21 (1-21), and the compound has obvious cytotoxicity on hepatic stellate cells and can be used for preparing anti-hepatic fibrosis drugs.
In order to achieve the above object of the present invention, the present invention provides the following technical solutions:
the invention provides a series of sesquiterpene dimer compounds artemzhongdianolides A-A21 (1-21), which have the structure shown in the following formula (I):
the invention provides a preparation method of the sesquiterpene dimer compound 1-21, which comprises the steps of crushing a dry upper part of Chinese mugwort, carrying out cold leaching extraction twice by using 95% ethanol for 4 days each time, combining ethanol extract, recovering ethanol extract under reduced pressure, dispersing the extract in water, extracting by using ethyl acetate, concentrating to obtain an ethyl acetate extract part, carrying out silica gel column chromatography on the ethyl acetate extract part, and carrying out gradient elution by using acetone-petroleum ether in terms of v/v, wherein the elution is carried out by using 0:100,5:95,10:90,20:80,40:60,50:50 and 100:0 as eluent to obtain seven fractions Fr.A-Fr.G; subjecting the fraction Fr.C to silica gel column chromatography, eluting with ethyl acetate-petroleum ether in terms of v/v, 5:95,10:90 and 20:80 to obtain five fractions Fr.C1-Fr.C5; the fraction fr.c5 was subjected to MCI gel CHP20P column chromatography, eluted with a water-methanol 10:90 and 0:100 gradient and semi-preparative HPLC to afford compound 1,2,3,4,5,6,9,10,11,12; subjecting the fraction Fr.D to MCI gel CHP20P column chromatography, and performing gradient elution with water-methanol 40:60, 60:40, 80:20 and 100:0 to obtain 4 fractions Fr.D1-Fr.D4; fr.D1 was eluted with a gradient of acetone-chloroform in v/v, 2:98, 10:90 and 20:80 by silica gel column chromatography followed by semi-preparative HPLC to give compound 7,8,13,14,15,16,17,18,19,20,21.
The invention provides application of the sesquiterpene dimer compounds 1-21 in preparing anti-hepatic fibrosis medicines. The method of the present invention is not particularly limited, and methods well known in the art may be used.
The invention provides a pharmaceutical composition, which comprises at least one of sesquiterpene dimer compounds 1-21 in the technical scheme and a pharmaceutically acceptable carrier or excipient.
And the application of the pharmaceutical composition in preparing anti-hepatic fibrosis medicines is also provided. And, the preparation method of the pharmaceutical composition, according to the preparation method of the compound, the compound 1-21 is prepared, then one or any combination of the compound 1-21 is taken, and a pharmaceutically acceptable carrier is added.
When at least one of the compounds 1 to 21 is used for the preparation of an anti-hepatic fibrosis drug, the present invention preferably uses the compound 1 to 21 directly or in the form of a pharmaceutical composition.
The invention provides a pharmaceutical composition comprising at least one of the above compounds 1-21 and a pharmaceutically acceptable carrier or excipient. In the present invention, the pharmaceutically acceptable carrier or excipient is preferably a solid, semi-solid or liquid diluent, filler and pharmaceutical preparation adjuvant. The pharmaceutically acceptable carrier or excipient is not particularly limited, and pharmaceutically acceptable carriers and/or excipients which are well known in the art, nontoxic and inert to human and animals can be selected.
The preparation method of the pharmaceutical composition is not particularly limited, at least one of the compounds 1-21 is directly mixed with a pharmaceutically acceptable carrier or excipient, the mixing process is not particularly limited, and the pharmaceutical composition can be obtained by selecting processes well known in the art.
The invention provides application of the pharmaceutical composition in preparation of anti-liver cancer drugs. The method of the present invention is not particularly limited, and methods well known in the art may be used.
In the present invention, when the pharmaceutical composition is used for preparing an anti-hepatic fibrosis drug, the content of the composition in the drug is preferably 0.1 to 99%; in the pharmaceutical composition, the content of at least one of the compounds 1 to 21 in the pharmaceutical composition is preferably 0.5 to 90%. The pharmaceutical composition of the present invention is preferably used in the form of a unit weight dose. In the present invention, the prepared medicine may be preferably administered in both injection (intravenous injection, intramuscular injection) and oral administration.
Compared with the prior art, the invention has the following advantages:
1. the invention provides a series of novel sesquiterpene dimer compounds, artemzhongdianolides A-A21 (1-21).
2. The invention provides a novel method for preparing novel compounds 1-21, which has the advantages of easily available raw materials, easy operation and suitability for industrial production.
3. The invention provides a pharmaceutical composition with novel compounds 1-21 as active ingredients, and provides a novel medicine with better medicinal effect for a novel anti-hepatic fibrosis medicine.
4. Compounds 1-21 of the present invention have potent cytotoxic activity against hepatic stellate cells (HSC-LX 2), and compounds 2,6 and 13 have potent cytotoxic activity against IC 50 The values are 22.1,24.3 and 14.0 mu M respectively, and the activity is 6-10 times of that of the positive drug silybin; compound 1,12,14,15,19,21 also exhibits a certain cytotoxic activity, IC thereof 50 The values are 32.5,95.2,84.2,69.4,34.5 and 46.9 mu M respectively, the activity is superior to that of the positive drug silybin, the other compounds have a certain HSC-LX2 cytotoxicity, and the inhibition rate is between 14.2 and 88.5 percent when the concentration is 400 mu g/mL.
Description of the drawings:
FIG. 1 is a structural formula of compounds 1-21 of the present invention;
FIG. 2 is a schematic diagram of the X-single crystal diffraction structure of Compound 1 of the present invention;
FIG. 3 is a schematic diagram of the X-single crystal diffraction structure of Compound 13;
FIG. 4 is a schematic diagram of the X-ray single crystal diffraction structure of compound 14.
The specific embodiment is as follows:
for a better understanding of the essence of the present invention, the sesquiterpene dimer compounds of the present invention, artemzhongdianolides A1-A21 (1-21), and methods for preparing the same, structural identification, pharmacological effects thereof will be further described with reference to the following test examples and examples of the present invention, but the present invention is not limited thereto.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
preparation of sesquiterpenoid dimer Compound according to the invention, artemzhongdianolides A1-A21 (1-21):
the dry upper part of the Chinese mugwort in 25kg is crushed, extracted twice with 95% ethanol at room temperature for 4 days each time by 200 liters, the ethanol extracts are combined, the organic solvent is recovered under reduced pressure to obtain crude extract, the extract is dispersed in water and then extracted with ethyl acetate, then the ethyl acetate extract is concentrated to obtain 749g of ethyl acetate part, then the ethyl acetate extract part is dissolved with ethyl acetate and adsorbed on 7.5kg of silica gel (200-300 meshes) for silica gel column chromatography, and seven fractions of Fr.A-Fr.G (155,94,117,79,163,66 and 52 g) are obtained by gradient elution with acetone-petroleum ether in v/v, 0:100,5:95,10:90,20:80,40:60,50:50 and 100:0 as eluent; fractions fr.c (117 g) were eluted with ethyl acetate-petroleum ether in v/v by silica gel column chromatography to give five fractions fr.c1-fr.c5 (10,27,37,20 and 16 g) at 5:95,10:90 and 20:80; fractions fr.c5 were subjected to column chromatography on silica gel with ethyl acetate-chloroform in v/v, 2:98, 10:90 and 20:80 to give 3 fractions C5.1-C5.3, fractions fr.c5.2 were subjected to column chromatography on MCI gel CHP20P with water-methanol 10:90 and 0:100 gradient elution to give fractions C5.2.1-C5.2.4, fractions fr.c5.2.2 were subjected to column chromatography on silica gel with ethyl acetate-chloroform in v/v, 2:98, 10:90 and 20:80 elution to give five fractions C5.2.2.1-C5.2.2.5, fraction C5.2.2.2 was subjected to semi-preparative liquid phase (H) 2 O-MeOH,35:65,3.0 mL/min) to give compound 3 (7 mg), fraction C5.2.2.3 was subjected to semi-preparative liquid phase (H 2 O-CH 3 CN,46:54,3.0 mL/min) to give compound 10 (5 mg), fraction C5.2.2.5 was subjected to semi-preparative liquid phase (H 2 O-CH 3 CN,50:50,3.0 mL/min) to give compound 11 (6 mg), flow C5.2.3 was subjected to gel column chromatography, preparative and semi-preparative liquid phases (H) 2 O-MeCN, 58:42) to give compound 1 (5 mg), 6 (2 mg), 9 (2 mg) and 12 (3 mg). Fraction C5.2.4 was eluted with ethyl acetate-chloroform in v/v from a silica gel column, fractions 2:98, 10:90 and 20:80 gave three fractions C5.2.4.1-5.2.4.3, fraction C5.2.4.2 was purified by gel column chromatography and semi-preparative liquid phase (H 2 O-MeCN 50:50 Compound 4 (3 mg) and 5 (2 mg) were obtained, and fraction C5.2.4.3 was subjected to semi-preparative liquid phase (H 2 O-MeOH,42:58,3.0 mL/min) afforded Compound 2 (4 mg). Subjecting the fraction Fr.D to MCI gel CHP20P column chromatography, and performing gradient elution with water-methanol 40:60, 60:40, 80:20 and 100:0 to obtain 4 fractions Fr.D1-Fr.D4; fr.D1 was eluted with acetone-chloroform in v/v gradient from 2:98, 10:90 and 20:80 to give five fractions D1.1-D1.5, fr.D1.5 was eluted with ethyl acetate-chloroform in v/v gradient from 2:98, 10:90 and 20:80 to give five fractions D1.5.1-D1.5.5, fraction D1.5.1 was subjected to semi-preparative liquid phase (H 2 O-MeOH,60:40,3.0 mL/min) to give compound 14 (128 mg), fraction D1.5.2 was subjected to gel column chromatography and semi-preparative liquid phase (H 2 O-MeCN 50:50) to give compounds 15 (12 mg), 18 (24 mg) and 17 (10 mg), fraction D1.5.4 was subjected to semi-preparative liquid phase (H 2 O-MeCN,75:25,3.0 mL/min) to give compound 21 (4 mg), fraction D1.5.5 was subjected to gel column chromatography and semi-preparative liquid phase (H 2 O-MeCN 55:45) to give compounds 7 (10 mg), 8 (6 mg) and 20 (4 mg), fr.D3 was eluted with a gradient of acetone-chloroform in v/v from 2:98, 10:90 and 20:80 by means of a silica gel column chromatography to give five fractions D3.1-D3.5, fr.D3.1 was eluted with a gradient of acetone-petroleum ether in v/v from 2:98, 10:90 and 20:80 by means of a silica gel column chromatography to give five fractions D3.1.1-D3.1.5, fraction D3.1.4 was eluted with a gradient of acetone-petroleum ether in v/v from 2:98, 10:90 and 20:80 by means of a gel column chromatography and semi-preparative liquid phase (H) 2 O-MeCN 50:50) to give compound 13 (10 mg), fraction D3.1.4 was subjected to semi-preparative liquid phase (H 2 O-MeCN 50:50) gave compounds 16 (2 mg) and 19 (4 mg).
Structural data for compounds 1-21:
melting point is adoptedX-4B micro-melting point apparatus (Shanghai precision scientific instruments Co., ltd.); specific optical rotation was determined by an Autopol VI polarimeter (Rudolph Research Analytical, hackettston, USA); infrared spectroscopy was performed by an ATR attenuated total reflectance-DIAMOND crystal (ATR ITX-DIAMOND) method using a NICOLET iS10 infrared spectrometer (Thermo Fisher Scientific, madison, USA); the UV spectrum was determined by UV-2401PC type UV spectrometer (Shimadzu, kyoto, japan); ECD spectra were obtained by Applied Photophysics round dichroscope (Applied Photoph)ysics, surrey, UK); nuclear magnetic resonance spectroscopy was determined using an Avance III 600 (Bruker, bremerhaven, germany) superconducting nuclear magnetic resonance apparatus using TMS (tetramethylsilane) as an internal standard; high resolution mass spectra were determined using Shimadzu LC-MS-IT-TOF (Shimadzu, kyoto, japan), agilent UPLC/Q-TOF and G6230 mass spectrometers (Agilent Technologies, santa Clara, USA); thin layer chromatography silica gel plate HSGF254 was purchased from nice river friends silica gel development limited; column chromatography silica gel (200-300 meshes) is produced by Yi Ling Shang Hai Xiang chemical industry Co., ltd; column chromatography sephadex LH-20 was purchased from GE Healthcare Bio-Sciences AB company; the high performance liquid chromatograph is purchased from Shimadzu corporation, the controller model is CBM-20A, the pump model is LC-20AR, the detector model is SPD-M20A, the column temperature box model is AT-350, the chromatographic column model is Agilent-Eclipse XDB-C18 (5 μm, 9.4X1250 mm); chromatographic pure acetonitrile was purchased from merida, and deionized channel water was purified by the mingchem-D24 UV Merk Millipore system; the medium-pressure liquid phase (Dr Flash-II) is a product of Shanghai Lisui corporation, mitsubishi corporation of Japan, MCI column, model CHP-20P (75-150 μm); analytically pure methanol and acetonitrile were purchased from the Tianjin metallocene chemical reagent plant; the color-developing agent is 10% H 2 SO 4 -EtOH solution.
Artemzhongdianolide A1(1)
The molecular formula: c (C) 31 H 38 O 8
Molecular weight: 538
Traits: colorless needle crystal
Optical rotation:(c 0.066, methanol)
HRESIMS (+) m/z Experimental values 539.2642[ M+H ]] + Calculated 539.2639[ M+H ]] + 。
IR v max :3441,1766,1716,1642,1458,1265,1063cm –1 。
1 H NMR 13 C NMR (DEPT) dataSee tables 1 and 4.
Artemzhongdianolide A2(2)
The molecular formula: c (C) 31 H 38 O 8
Molecular weight: 538
Traits: white powder
Optical rotation:(c 0.050, methanol)
HRESIMS (+) m/z Experimental values 539.2626[ M+H ]] + Calculated 539.2639[ M+H ]] + 。
IR v max :3442,1755,1705,1633,1457,1384,1082cm -1 。
ECD (methanol) lambda max (Δε):197(+11.4),235(+16.4),247(–17.2),326(+2.2)nm。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 1 and 4.
Artemzhongdianolide A3(3)
The molecular formula: c (C) 30 H 36 O 8
Molecular weight: 524
Traits: white powder
Optical rotation:(c 0.056, methanol)
HRESIMS (+) m/z, experimental value 525.2490[ M+H ]] + Calculated 525.2483[ M+H ]] + 。
IR v max :3441,1769,1745,1706,1646,1378,1262,1212,1128cm -1 。
ECD (methanol) lambda max (Δε):219(–8.0),235(+7.9),320(–2.4)nm。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 1 and 4.
Artemzhongdianolide A4(4)
The molecular formula: c (C) 30 H 38 O 8
Molecular weight: 526
Traits: white powder
Optical rotation:(c 0.010, methanol)
HRESIMS (-) m/z Experimental values 525.2506[ M-H ]] – Calculated 525.2494[ M-H ]] – 。
IR v max :3429,1753,1628,1457,1377,1264,1158,1064cm -1 。
ECD (methanol) lambda max (Δε):197(+11.2),216(+1.6)nm。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 1 and 4.
Artemzhongdianolide A5(5)
The molecular formula: c (C) 31 H 40 O 8
Molecular weight: 540
Traits: white powder
Optical rotation:(c 0.030, methanol)
HRESIMS (+) m/z Experimental values 585.2699[ M+HCOO ]] – Calculated 585.2705[ M+HCOO] – 。
IR v max :3441,1755,1633,1457,1384,1262,1085cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 1 and 4.
Artemzhongdianolide A6(6)
The molecular formula: c (C) 31 H 40 O 9
Molecular weight: 556
Traits: white powder
Optical rotation:(c 0.10, methanol)
HRESIMS (+) m/z Experimental values 557.2731[ M+H ]] + Calculated 557.2745[ M+H ]] + 。
IR v max :3440,1765,1720,1634,1458,1383,1263,1087,872cm –1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 1 and 4.
Artemzhongdianolide A7(7)
The molecular formula: c (C) 30 H 38 O 8
Molecular weight: 526
Traits: white powder
Optical rotation:(c 0.052, methanol)
HRESIMS (+) m/z, experimental value 525.2489[ M-H ]] – Calculated 525.2494[ M-H ]] – 。
IR v max :3434,1753,1636,1456,1380,1321,1145,994cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 1 and 4.
Artemzhongdianolide A8(8)
The molecular formula: c (C) 30 H 36 O 8
Molecular weight: 524
Traits: white powder
Optical rotation:(c 0.053, methanol)
HRESIMS (+) m/z Experimental value 523.2328[ M-H ]] – Calculated 523.2337[ M-H ]] – 。
IR v max :3435,1758,1713,1635,1456,1379,1264,1135cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 2 and 4.
Artemzhongdianolide A9(9)
The molecular formula: c (C) 30 H 36 O 8
Molecular weight: 524
Traits: white powder
Optical rotation:(c 0.030, methanol)
HRESIMS (+) m/z Experimental values 525.2482[ M+H ]] + Calculated 525.2483[ M+H ]] + 。
IR v max :3433,1763,1633,1384,1262,1126,1026,892cm -1 。
1 H NMR and 13 the C NMR (DEPT) data are shown in tables 2 and 4.
Artemzhongdianolide A10(10)
The molecular formula: c (C) 30 H 38 O 8
Molecular weight: 526
Traits: white powder
Optical rotation:(c 0.020, methanol)
HRESIMS (-) m/z Experimental value 527.2627[ M+H ]] + Calculated 527.2639[ M+H ]] + 。
IR v max :3436,1751,1633,1457,1264,1157,1070cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 2 and 4.
Artemzhongdianolide A11(11)
The molecular formula: c (C) 30 H 38 O 9
Molecular weight: 542
Traits: white powder
Optical rotation:(c 0.050, methanol)
HRESIMS (+) m/z Experimental value 565.2418[ M+Na ]] + Calculated 565.2408[ M+Na] + 。
IR v max :3435,1751,1630,1457,1321,1264,1158,1068,852cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 2 and 5.
Artemzhongdianolide A12(12)
The molecular formula: c (C) 31 H 40 O 9
Molecular weight: 556
Traits: colorless powder
Optical rotation:(c 0.102, methanol)
HRESIMS (+) m/z Experimental values 579.2566[ M+Na ]] + Calculated 579.2565[ M+Na ]] + 。
IR v max :3440,1752,1632,1457,1371,1262,1158,1048,851cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 2 and 5.
Artemzhongdianolide A13(13)
The molecular formula: c (C) 30 H 36 O 6
Molecular weight: 492
Traits: colorless needle crystal
Optical rotation:(c 0.060, methanol)
HRESIMS (+) m/z Experimental values 493.2591[ M+H ]] + Calculated 493.2585[ M+H ]] + 。
IR v max :3510,1766,1703,1612,1318,1218,1125,1026cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 2 and 5.
Artemzhongdianolide A14(14)
The molecular formula: c (C) 30 H 36 O 8
Molecular weight: 524
Traits: colorless needle crystal
Optical rotation:(c 0.058, methanol)
HRESIMS (+) m/z Experimental value 523.2334[ M-H ]] – Calculated 523.2337[ M-H ]] – 。
IR v max :3444,1759,1708,1644,1456,1154,1045cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 2 and 5.
Artemzhongdianolide A15(15)
The molecular formula: c (C) 30 H 36 O 6
Molecular weight: 524
Traits: white powder
Optical rotation:(c 0.060, methanol)
HRESIMS (+) m/z, experimental value 525.2492[ M+H ]] + Calculated 525.2483[ M+H ]] + 。
IR v max :3435,1753,1707,1455,1243,1155,1018cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 3 and 5.
Artemzhongdianolide A16(16)
The molecular formula: c (C) 31 H 41 O 9
Molecular weight: 556
Traits: white powder
Optical rotation:(c 0.050, methanol)
HRESIMS (+) m/z Experimental values 557.2751[ M+H ]] + Calculated 557.2745[ M+H ]] + 。
IR v max :3437,1756,1630,1458,1329,1244,1037,875cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 3 and 5.
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Artemzhongdianolide A17(17)
The molecular formula: c (C) 30 H 38 O 8
Molecular weight: 526
Traits: white powder
Optical rotation:(c 0.050, methanol)
HRESIMS (+) m/z experimental value 549.2465[ M+Na ]] + Calculated 549.2459[ M+Na] + 。
IR v max :3429,1749,1632,1458,1323,1155,896cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 3 and 5.
Artemzhongdianolide A18(18)
The molecular formula: c (C) 30 H 38 O 9
Molecular weight: 542
Traits: white powder
Optical rotation:(c 0.080, methanol)
HRESIMS (+) m/z Experimental value 565.2410[ M+Na ]] + Calculated 565.2408[ M+Na] + 。
IR v max :3436,1747,1634,1322,1155,984,851cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 3 and 5.
Artemzhongdianolide A19(19)
The molecular formula: c (C) 31 H 40 O 9
Molecular weight: 556
Traits: colorless needle crystal
Optical rotation:(c 0.040, methanol)/(methanol)>
HRESIMS (+) m/z Experimental values 555.2594[ M-H ]] – Calculated 555.2600[ M-H ]] – 。
IR v max :3435,1748,1630,1457,1264,1194,1079cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 3 and 5.
Artemzhongdianolide A20(20)
The molecular formula: c (C) 30 H 38 O 8
Molecular weight: 526
Traits: white powder
Optical rotation:(c 0.044, methanol)
HRESIMS (+) m/z experimental value 549.2466[ M+Na ]] + Calculated 549.2459[ M+Na] + 。
IR v max :3435,1749,1636,1455,1155,1018cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 3 and 5.
Artemzhongdianolide A21(21)
The molecular formula: c (C) 30 H 36 O 8
Molecular weight: 524
Traits: white powder
Optical rotation:(c 0.020, methanol)
HRESIMS (+) m/z, experimental value 525.2479[ M+H ]] + Calculated 525.2483[ M+H ]] + 。
IR v max :3436,1757,1628,1321,1268,1015cm -1 。
1 H NMR 13 The C NMR (DEPT) data are shown in tables 3 and 5.
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Example 2:
cytotoxic activity of sesquiterpenoid dimer compound artemzhongdianolides A-A21 (1-21) on HSC-LX 2.
1. Materials and methods
1.1 materials
Human hepatic stellate cell line (hepatic stellate cells line LX, HSC-LX 2) was purchased from Shanghai Ji Ning Biotechnology Co., ltd; RPMI-1640 medium and fetal bovine serum were purchased from Gibco BRL (NY, USA); MTT was purchased from the tsu biotechnology company, racing state, inc.
1.2 instruments
Flex Station 3 bench-top multifunctional microplate reader (Bio-RAD 680, USA); analytical balances (AG 135, metler Toledo, china); incubator (DHP-9082, shanghai).
1.3 Experimental procedure
The toxic activity of the samples on HSC-LX2 cells was determined using the MTT assay. HSC-LX2 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Cells grown in log phase were taken at 1X 10 4 The density of the holes is inoculated in a 96-well plate, the maintenance solution is replaced by a culture medium containing test samples with different concentrations after 24 hours, a cell control group only added with the maintenance solution is arranged at the same time, and silybin is used as positive medicine control. After 48h of incubation, the broth was discarded and 100. Mu.L of MTT solution (1 mg/mL) was added; incubating for 4 hours at 37 ℃, discarding the MTT solution and adding 100 mu L of DMSO to dissolve the crystals of alpha-fetid; finally, absorbance values were measured at 490nm with a microplate reader. HSC-LX2 cell inhibition rate calculation formula is inhibition rate (%) = [ A (blank) -A (sample)]A (blank) ×100%. Its half partNumber inhibition concentration (50%inhibitory concentration,IC) 50 ) Calculation was performed using Graphpad Prism 5 software.
2. Results
All compounds isolated were evaluated for their HSC-LX2 cytotoxic activity in vitro (Table 5). Compounds 1-21 have potent cytotoxic activity against hepatic stellate cells (HSC-LX 2), and compounds 2,6 and 13 have potent cytotoxic activity against IC 50 The values are 22.1,24.3 and 14.0 mu M respectively, and the activity is 6-10 times of that of the positive drug silybin; compound 1,12,14,15,19,21 also exhibits a certain cytotoxic activity, IC thereof 50 The values are 32.5,95.2,84.2,69.4,34.5 and 46.9 mu M respectively, the activity is superior to that of the positive drug silybin, the other compounds have a certain HSC-LX2 cytotoxicity, and the inhibition rate is between 14.2 and 88.5 percent when the concentration is 400 mu g/mL.
TABLE 5 HSC-LX2 cytotoxic Activity of Compounds
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Inhibition rate and IC 50 All from three independent experiments.
3. Conclusion(s)
Experimental results show that compounds 1-21 have strong cytotoxic activity against hepatic stellate cells (HSC-LX 2), and that compounds 2,6 and 13 have strong cytotoxic activity against IC 50 The values are 22.1,24.3 and 14.0 mu M respectively, and the activity is 6-10 times of that of the positive drug silybin; compound 1,12,14,15,19,21 also exhibits a certain cytotoxic activity, IC thereof 50 The values are 32.5,95.2,84.2,69.4,34.5 and 46.9 mu M respectively, the activity is superior to that of the positive drug silybin, the other compounds have certain HSC-LX2 cytotoxicity, and the inhibition rate is between 14.2 and 88.5 percent when the concentration is 400 mu g/mL
The results show that the sesquiterpene dimer compound in the Chinese mugwort, artemzhongdianolides A-A21 (1-21), can be used as a medicament for preparing medicaments for resisting hepatic fibrosis diseases.
Formulation examples 1-7:
in the following formulation examples, conventional reagents are selected and formulation preparation is performed according to the conventional methods, and this application example only embodies that at least one of the compounds 1 to 21 of the present invention can be prepared into different formulations, and specific reagents and operations are not particularly limited:
1. dissolving at least one of the compounds 1-21 with DMSO, adding water for injection according to conventional method, fine filtering, packaging, and sterilizing to obtain injection with concentration of 0.5-5mg/mL.
2. Dissolving at least one of the compounds 1-21 with DMSO, dissolving in sterile injectable water, stirring to dissolve, filtering with sterile suction filter funnel, sterile fine filtering, packaging in ampoule, freeze drying at low temperature, and sealing under sterile condition to obtain powder for injection.
3. Adding excipient into at least one of the compounds 1-21 according to the mass ratio of the compound to the excipient of 9:1, and preparing into powder.
4. At least one of the compounds 1-21 is added with excipient according to the mass ratio of 5:1, and the mixture is granulated and tabletted.
5. At least one of the compounds 1-21 is prepared into oral liquid according to the conventional oral liquid preparation method.
6. Adding excipient into at least one of the compounds 1-21 according to the mass ratio of 5:1, and making into capsule.
7. Adding excipient into at least one of the compounds 1-21 according to the mass ratio of the compound to the excipient of 5:1, and making into granule.
The above examples show that the present invention provides a compound of Zhongdian mugwort, its preparation method and application, pharmaceutical composition and its application. The 21 novel sesquiterpenoids provided by the invention have different degrees of cytotoxicity on human hepatic stellate cells HSC-LX2, can be combined with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition, and can be used for preparing anti-hepatic fibrosis drugs.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (6)
1. Sesquiterpene dimer compounds 1-2, 6, 12-15, 19,21 shown in the following structural formula,
2. a process for the preparation of sesquiterpene dimer compounds 1-2, 6, 12-15, 19,21 according to the structural formula of claim 1, characterized in that the process comprises the steps of: pulverizing dry upper part of Zhongdian moxa, cold-leaching with 95% ethanol twice for 4 days each time, mixing ethanol extracts, recovering ethanol extract under reduced pressure, dispersing the extract in water, extracting with ethyl acetate, concentrating to obtain ethyl acetate extract, subjecting the ethyl acetate extract to silica gel column chromatography, and gradient eluting with acetone-petroleum ether (v/v) as eluent, wherein the eluent is 0:100,5:95,10:90,20:80,40:60,50:50 and 100:0 to obtain seven fractions Fr.A-Fr.G; subjecting the fraction Fr.C to silica gel column chromatography, eluting with ethyl acetate-petroleum ether in terms of v/v, 5:95,10:90 and 20:80 to obtain five fractions Fr.C1-Fr.C5; the fraction fr.c5 was subjected to MCI gel CHP20P column chromatography, eluted with a water-methanol 10:90 and 0:100 gradient and semi-preparative HPLC to afford compound 1,2,6,12; subjecting the fraction Fr.D to MCI gel CHP20P column chromatography, and performing gradient elution with water-methanol 40:60, 60:40, 80:20 and 100:0 to obtain 4 fractions Fr.D1-Fr.D4; fr.D1 was eluted with a gradient of acetone-chloroform in v/v, 2:98, 10:90 and 20:80 by silica gel column chromatography followed by semi-preparative HPLC to give compound 13,14,15,19,21.
3. Use of sesquiterpene dimer compounds 1-2, 6, 12-15, 19,21 of the structural formula as defined in claim 1 for the preparation of an anti-hepatic fibrosis medicament.
4. A pharmaceutical composition comprising at least one of the compounds 1-2, 6, 12-15, 19,21 of the structural formula of claim 1 and a pharmaceutically acceptable carrier.
5. The use of the pharmaceutical composition of claim 4 for the preparation of an anti-liver fibrosis medicament.
6. A process for the preparation of a pharmaceutical composition according to claim 4, comprising the steps of: pulverizing dry upper part of Zhongdian moxa, cold-leaching with 95% ethanol twice for 4 days each time, mixing ethanol extracts, recovering ethanol extract under reduced pressure, dispersing the extract in water, extracting with ethyl acetate, concentrating to obtain ethyl acetate extract, subjecting the ethyl acetate extract to silica gel column chromatography, and gradient eluting with acetone-petroleum ether (v/v) as eluent, wherein the eluent is 0:100,5:95,10:90,20:80,40:60,50:50 and 100:0 to obtain seven fractions Fr.A-Fr.G; subjecting the fraction Fr.C to silica gel column chromatography, eluting with ethyl acetate-petroleum ether in terms of v/v, 5:95,10:90 and 20:80 to obtain five fractions Fr.C1-Fr.C5; the fraction fr.c5 was subjected to MCI gel CHP20P column chromatography, eluted with a water-methanol 10:90 and 0:100 gradient and semi-preparative HPLC to afford compound 1,2,6,12; subjecting the fraction Fr.D to MCI gel CHP20P column chromatography, and performing gradient elution with water-methanol 40:60, 60:40, 80:20 and 100:0 to obtain 4 fractions Fr.D1-Fr.D4; subjecting Fr.D1 to silica gel column chromatography, eluting with acetone-chloroform in v/v, gradient of 2:98, 10:90 and 20:80, and semi-preparative HPLC to obtain compound 13,14,15,19,21; then one or any combination of the compounds is taken, and a pharmaceutically acceptable carrier is added.
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